bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423411; this version posted December 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Dominant Remodeling of Cattle Rumen Microbiome by Schedonorus arundinaceus (Tall 2 Fescue) KY-31 Carrying a Fungal Endophyte 3 4 Bela Haifa Khairunisa1, Dwi Susanti2#a, Usha Loganathan2, Christopher D. Teutsch3#b, Brian T. 5 Campbell3#c, David Fiske4†, Carol A. Wilkinson3, Frank O. Aylward5, Biswarup Mukhopadhyay2* 6 7 8 1Genetics, Bioinformatics, and Computational Biology, Virginia Tech, Blacksburg, VA, USA 9 2Department of Biochemistry, Virginia Tech, Blacksburg, VA, USA 10 3Southern Piedmont Agricultural Research and Extension Center, Virginia Tech, Blackstone, VA, 11 USA 12 4Shennandoah Valley Agricultural Research and Extension Center, Virginia Tech, Raphine, VA, 13 USA 14 5Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA. 15 #aCurrent address: Elanco Animal Health, Greenfield, IN, USA. 16 #bCurrent address: University of Kentucky Research and Education Center, Princeton, KY, USA. 17 #cPresent address: Archer Daniels Midland Company, Decatur, IL, USA. 18 19 20 *Corresponding Author 21 E-mail: [email protected] 22 23 † Deceased 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423411; this version posted December 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Abstract 25 Tall fescue KY-31 feeds ~20% of the beef cattle in the United States. It carries a fungal endophyte 26 that produces ergovaline, which causes toxicosis in cattle, leading to $2 billion revenue loss 27 annually. The MaxQ cultivar of the grass is non-toxic, but less attractive economically. To develop 28 ways of mitigating the toxicity, the rumen microbiome of cattle consuming KY-31 and MaxQ have 29 been analyzed, principally for identifying ergovaline transforming microorganisms and often using 30 fecal microbiome as a surrogate. We have hypothesized that KY-31 not only causes toxicosis, but 31 also impacts rumen metabolism broadly, and tested the hypothesis by analyzing rumen 32 microbiome compositions of cattle that grazed MaxQ with an intervening KY-31 grazing period 33 with 16S rRNA-V4 element as identifier. We found that KY-31 remodeled the cellulolytic and 34 saccharolytic communities substantially. This effect was not evident at whole microbiome levels 35 but in the compositions of sessile and planktonic fractions. A move from MaxQ to KY-31 lowered 36 the Firmicutes abundance in the sessile fraction and increased it in planktonic part and caused an 37 opposite effect for Bacteroidetes, although the total abundances of these dominant rumen 38 organisms remained unchanged. In the sessile fraction, the abundances of Fibrobacter, which 39 degrades less degradable fibers, and certain cellulolytic Firmicutes such as Pseudobutyrivibrio and 40 Butyrivibrio 2, dropped, and these losses were apparently compensated by increased occurrences 41 of Eubacterium and specific Ruminococcaceae and Lachnospiraceae. In planktonic fraction the 42 Tenericutes’ abundance increased as saccharolytic Bacteroidetes’ level dropped. Several potential 43 ergovaline degraders were enriched. A return to MaxQ restored the original Firmicutes and 44 Bacteroidetes distributions. However, the Fibrobacter and Butyrivibrio 2 abundances remained 45 low and their substitutes maintained significant presence. The rumen microbiome was influenced 46 minimally by animals’ fescue toxicosis and was distinct from previously reported fecal 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423411; this version posted December 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 47 microbiomes in composition. In summary, KY-31 and MaxQ cultivars of tall fescue were digested 48 in the cattle rumen with distinct consortia and the KY-31-specific features were dominant. The 49 study highlighted the importance of analyzing sessile and planktonic fractions separately. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423411; this version posted December 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Introduction 50 In the foregut or rumen of ruminants such as cattle, a complex microbial community 51 anaerobically degrades the feed, generating volatile fatty acids as carbon and energy nutrition for 52 the animals (1). The process also makes livestock major emitters of methane, a potent greenhouse 53 gas (1). For near 100 years there has been intense research focused on rumen microbiome 54 metabolism because of this importance, helping not only to optimize the feed utilization 55 efficiencies in economically important ruminants but also to develop microbial processes for 56 bioenergy production (1-3). In this backdrop, a lack of detailed knowledge of rumen microbiome 57 metabolism in cattle raised on tall fescue is a major gap as it impedes the efforts to utilize this 58 economic grass efficiently for beef and dairy production, to assess the associated impacts on 59 methane emission and for mining this source for novel biocatalysts. 60 Tall fescue (Schedonorus arundinaceus (Schreb.) Dumort., nom. cons. tall fescue) is one 61 of the primary perennial cool season forages that feeds up to 20% of beef cattle in the United States 62 (4). It is grown on more than 35 million acres in the transition zone of Southeastern US, known as 63 the fescue belt (5). In Argentina, Uruguay, and Australia, this forage is grown on over 8.65, 1.24 64 and 2.72 million acres of the pastures, respectively (6-8). The most widely used variety of the grass 65 is KY-31 and it is popular for its resilience towards pests and poor environmental conditions (5). 66 The resilience of KY-31 is due in part to its symbiotic interaction with a fungal endophyte, 67 Epichloë coenophiala (9). The fungus secretes a variety of ergot alkaloids that are beneficial to the 68 grass (9). However, one of these compounds, ergovaline, is toxic to the animals causing Tall 69 Fescue Toxicosis syndrome (10), which results in $2 billion of annual revenue loss for the US beef 70 and dairy industries (11, 12). The use of engineered varieties of tall fescue that either carry a E. 71 coenophiala strain that does not produce ergovaline (MaxQ) or are free of the endophyte (KY32, 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423411; this version posted December 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 72 Cajun I, and Bronson) have been promoted as a solution to this problem (9). However, these 73 varieties are less attractive economically due to higher costs for the seeds and pasture management, 74 and KY-31 remains widely used in the fescue belt (13, 14). The selection of fescue toxicosis 75 resistant or tolerant cattle remains an underdeveloped option (5, 15). All these efforts do not 76 consider the possibility that the rumen microbes instigate and could mitigate the above-mentioned 77 toxicity, although there are indications of such possibilities. In vitro studies suggest that certain 78 rumen microorganisms transform ergovaline to lysergic acid (16) and it is thought that lysergic 79 acid enters the blood stream of the cattle causing fescue toxicosis (16). The addition of isoflavone 80 producing grasses to a tall fescue diet reduces the severity of fescue toxicosis, and it is thought that 81 isoflavones suppresses ergovaline transforming microbes (11); the mechanism of this suppression 82 is unknown. In search of a microbial basis for the promotion and/or suppression of KY-31 toxicity, 83 the characteristics of the fecal microbiomes of cattle raised on KY-31 and a non-toxic tall fescue 84 have been compared, resulting in the identification of candidate microorganisms (17, 18). 85 Recently, the rumen microbiome of pregnant ewes grazing tall fescue with high and moderate 86 endophyte infection levels in parallel, were analyzed (19). However, these findings do not truly 87 represent the events occurring in the rumen for the following reasons. The feces and rumen harbor 88 distinct microbiomes, sharing ~30% of the species (17, 20). The study with the ewes involved the 89 analysis of rumen fluid drawn via orogastric tube insertion. and therefore, concerned only the 90 planktonic segment and not the whole microbiome as the sessile microbial population was not 91 targeted. We hypothesized that the differences between the microbial systems that KY-31 and 92 MaxQ enrich in cattle rumen are likely broader in nature, going beyond the transformation of 93 ergovaline and covering the overall degradation process occurring both in the planktonic and 94 sessile fractions. Accordingly, we have analyzed the composition of the sessile and planktonic 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423411; this version posted December 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.